Asier Sáez-Cirión, PhD Unité HIV Inflammation et Persistance Institut Pasteur A look at HIV‐1 journey inside the cell
Asier Sáez-Cirión, PhD Unité HIV Inflammation et Persistance
Institut Pasteur
A look at HIV‐1 journey inside the cell
Source: Monsef Benkirane
Source: NIH USA, redrawn by Carl Henderson
HIV-1 virion and genome organization
Structural and regulatory proteins:essential for viral replication
Accessory proteins: important determinants of virulence due to their ability to manipulate the host cell physiology for the benefit of the virus
Different steps that are targeted byantiretroviral molecules
Fusion inhibitors
Reverse transcription inhibitors
Integration inhibitors
Proteaseinhibitors
HIV‐1 replication depends on a complex interaction between viral and cellular factors
Adapted from Rambaut et al Nat Rev Gen 2004
HIV infects cells from the immune system, whichcontribute to its dissemination and persistance
T cells (47,CCR6+)macrophages
T cells
Microglial cells
Follicular DCsT Follicular Helper
hematopoietic progenitor cells?
CD4+ T lymphocytes Monocytes/macrophagesDendritic cells
However the virus does not replicate similarlyin these cells
The dynamics of viral replication are extremely different in T cells and myeloid cells.
Slow replication in myeloid cells has beenlinked to: Larger cytoplasm Decreased pool of dNTPs
CD4+ T cells
Macrophages
Dendriticcells
Saez‐Cirion et al unpublished
Different susceptibility of myeloid cells to HIV‐1
Monocytes
Dendritic cells Macrophages
Immature Mature Non activated Activated
HIV Resistant
HIV‐1 PermissiveHIV‐1 moderatelyPermissive
HIV Resistant HIV Resistant
Finzi et al. Cells 1997
The type of the cell HIV is infecting is going to determine its persistence
Cell susceptibility to infection
Establishment of HIV reservoir is a multifaceted process
Restriction factors
APOBEC3 proteinsTRIM5α proteinsBST‐2/TetherinSAMHD1MX2p21Others (?)
Dependence factors
Brass et al Science 2008
NIAID
Cell susceptibility to infectionSurvival to HIV infection
Resistance to HIV induced apoptosisEscape immune surveillance
Establishment of HIV reservoir is a multifaceted process
CTL
Finzi et al. Cells 1997
Cell susceptibility to infectionSurvival to HIV infection
Resistance to HIV induced apoptosisEscape immune surveillance
Cell Persistence
Half lifeTurnover
Establishment of HIV reservoir is a multifaceted process
HIV replication and the balance between restriction and dependence factors
Restriction factors
APOBEC3 proteinsTRIM5α proteinsBST‐2/TetherinSAMHD1MX2p21Others (?)
Dependence factors
Brass et al Science 2008
IFN HIV restriction factors
Ѵ Apolipoprotein B mRNA‐editing, enzyme‐catalytic polypeptide‐like 3
APOBEC3
Ѵ Bone marrow stromal cell antigen 2BST‐2/Tetherin
Ѵ Tripartite motif proteinsTRIM
SAM domain and HD domain 1SAMHD1
Ѵ Myxovirus resistance B MxB
Ѵ IFN‐induced transmembrane proteinsIFITMs
From Harris R S et al. J. Biol. Chem. 2012;287:40875-40883©2012 by American Society for Biochemistry and Molecular Biology
Sheehy et al.,2002; Conticelloetal.,2003; Harrisetal.,2003; Mangeat et al.,2003;Marianietal.,2003
HIV restriction by APOBEC3GAPOBEC3G interferes with reverse transcription by inducing numerous
deoxycytidine to deoxyuridine mutations in the negative strand of the HIV DNA
Hattlmann Mol Biol Int 2012Barr et al Plos Pathogens. 2008Kajaste‐Rudnitski et al J Virol. 2011Ghezzi et al AIDS 2013
TRIM22
Stremlau et al Nature 2004, PNAS 2006Sayah et al Nature 2004Pertel et al Nature 2011Ganser‐Pornillos et al PNAS 2011Mandell et al Dev Cell 2014
TRIM5αProteasomeAutophagy
Diaz‐Griffero F, Viruses 2011
Allouch et al CHM 2011
TRIM28
TRIM28
HIV restriction by TRIM proteinsTRIM5 present in cytoplasmic bodies recognizes motifs within the capsid proteins and interferes with the uncoating process, preventing successful reverse transcription and transport to the nucleus of the viral genome
HIV restriction by MxB
MxB and HIV‐1 Multitropic or ill‐defined mechanism?
Haller et al Trends in Immunology 2015
The Mx dynamin‐like GTPases are key antiviral effector proteins of the type I and type III interferon (IFN) systems
AP1
From Harris R S et al. J. Biol. Chem. 2012;287:40875-40883
Neil et al, 2008; Van Damme et al, 2008; Perez‐Caballero et al, 2009
HIV restriction by Tetherin/BST‐2
Neil, S.J., Zang, T., and Bieniasz, P.D. 2008. Nature 451:425–430.
Tetherin acts to physically tether budding virions to the cell surface of productively infected cells
Tetherin/BST‐2 as an exemple of species adaptation to host anti‐viral factors
Sauter Frontiers in Microbiology 2014
Related viruses have devised different means to counteract these factors
Uncoatingand reverse transcription
Entry and earlypost‐entry blocks
Exit block
Adapted from Suzuki and CraigieNature Reviews Microbiology 2007
TRIM5α
APOBEC3G
H. G. Göttlinger, Nature 2008
Tetherin
Vif
Ca
Vpu
IFITMNuclearentry
MxB
Fusion
SAMHD1
Vpx
HIV restriction factors
SAMHD1True barrier to HIV replication in non cycling cells
0.01
0.1
1
10
100
1000
10 14 17
p27 (ng/ml)
Days post‐infection
Vpx WTDvpx
HIV‐2Gh‐1
Vpx is requiredfor HIV‐2 and SIVmacreverse transcription
in macrophages
But not essential for HIV‐1 0,01
0,1
1
10
100
1000
4 8 12 15Days post‐infection
p24 (ng/ml)
HIV‐1BaL
Monsef Benkirane
From Harris R S et al. J. Biol. Chem. 2012
Lahouassa H et al. Nat Immunol. 2012
SAMHD1 two possible mechanisms:Degradation of the dNTP pool, RNAse activity
Lim and Emmerman. Nature 2011
SAMHD1 responsible for the block in resting CD4+ T cells and reduced susceptibility in myeloid cells
Descours et al, Retroviroly 2012
Cribier A. et al. Cell Reports, 2013
Phosphorylation of SAMHD1 is critical for its anti‐HIV activity
Benkirane et al
• p21: cyclin‐dependent kinase inhibitor (CDKI) (Harper J. Cell, 1993, Xiong Y, Nature, 1993).
• p21 plays a critical role in the control of cell growth (Sherr CJ & Roberts JM Genes & development, 1995).
• p21 plays a role in monocyte differentiation and survival (Xaus J, Immunity, 1999, Asada M, EMBO J, 1999)
p21Waf1/Cip1 a metabolic barrier to HIV replication
FcR
Bergamaschi et al., J Virol, 2009
Perez‐Bercoff et al., J Virol, 2003, David et al., J Immunol, 2006
0
0.5
1
1.5
2
2.5
3
US IVIg
p21m
RNA (Fold chan
ge)
p21 expressionBioluminescence Luciferase
Late RT
Copies per 100ng
DNA
0
1000
2000
3000
4000
5000
6000
7000
Ctrl si‐p21 si‐Neg
USS
p21 block HIV‐1 reverse transcription in macrophages
FcR
FcR
IC
p21 knockdowns intracellular dNTP levels by interfering with their synthesis via E2F1 and RNR2
Allouch A. et al., PNAS, 2013
dATP
Numerous cell factors pave the way to HIV infection
Brass et al Science 2008
Entry: surface receptors critical also for post‐entry events of viral replication
Cicala PNAS 2005Sodhi et al Nat Rev Mol Cell Biol 2004
R5 and X4 HIV envelopes induce distinct gene expression profiles
Arhel et al. Nat Methods
Intracellular trafficking: moving swiftly to the nucleus
McDonald JCB 2002
HIV‐1 depends on microtubules and actin filaments to move inside the cell
Model for HIV‐1 integration site selection.After entry into the nucleus through thenuclear pore, the viral DNA integrates into theactive chromatin closest to the
HIV‐1 integration occurs close to the nuclear pore
Marini et al Nature 2015
LEDGF/p75 acts as a chromatin docking factorLEDGF/p75 tethers HIV integrase tochromatin, protects it from degradation,and strongly influences the pattern of HIVintegration
LEDGF/p75 may repress HIV expression inlatently infected cells by maintaininghistone occupancy at the HIV promote
Gerard et al Cell Host Microbe 2015
Sloan and Wainberg Retrovirology 2011 8:52 doi:10.1186/1742‐4690‐8‐52
Multiple HIV DNA forms within the nucleusTranscription from preintegrated or unintegrated DNA may contribute to modify cell environment
Determinants of HIV-1 latency
P‐TEFb
HIV transcription requires the coalition of multiple factors
Van Lint et al Retrovirology 2013
‐ The site of integration
‐ The pool of transcriptionfactors
‐ Chromatin organization
‐ Tat and tat associatedfactors
L. Colin et al Retrovirology 2009
Several mechanisms can contribute to HIV latency
Pomeranz science 2009
Pomeranz science 2009
Chen et al PNAS 2014
Non coding RNAs can block HIV after transcription
HIV hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus
release from infected cells.
HIV assembly ESCRT and… lipids HIV buds from rigid lipid domains and requires cholesterol rich environments
Balasubramaniam and Freed, Physiology 2011
Bernardino de la Serna
Saad et al PNAS 2006
HIV budding can occur at different compartments and this may vary depending on the cell type
Balasubramaniam and Freed, Physiology 2011
How does all this translate in vivo?
Protection against infectionHIV‐1 Highly Exposed
Seronegative
Control of infectionHIV‐1/SIV controllers
Study of natural mechanisms of control of HIV‐1 infection
Intravenous drugusers
Risk factor
Needel sharing-drugabuse: 13-31 years
Serology:HBV: 82% HCV: 100%HTLV:80%
Studies in highly exposed seronegativeindividuals
Saez‐Cirion A. et al, Retrovirology 2006
1‐Restriction of R5 viruses linked to:a) CCR5 heterozygous mutations and downregulation of the co‐receptorb) enhanced sensitivity to ‐chemokines
2‐Pantropic restriction of HIV‐1 and other retroviruses during reverse transcription
Three persistent HIV‐1 restriction mechanisms
identified in the CD4 T cells
Drug development Cure strategies
Time
Progressor
Plas
ma
Vir
al lo
ad
HIC
HIV controllers (HIC): cART naïve infected individualsspontaneously controlling HIV-1 infection for long
periods of time
Lambotte O et al, Clin Infect Dis. 2005Buffassa et al, PLoS One 2011
Since 2009: ANRS CO18 CO21 cohort
~200 HIC <0.5 % of HIV infected patients
CD4+ T cells and macrophages from HIC have reduced susceptibility to HIV‐1 infection
See also Chen et al JCI 2011, Buzon et al JVI 2011; Graf et al PLoS Path 2011
Sáez-Cirión et al Blood 2011
Intrinsic cell resistance may contribute to limit the size of HIV reservoir in HIC
HIC HD VIRp24 in culture sup
erna
tants (ng
/ml)
10‐1
100
101
102
103
104
p<0.00001
p=0.017
CD4+ T cells from HIC have been shown to express high levels of p21 and APOBEC3G
Time
Progressor
Plas
ma
Vir
al lo
ad
Natural or induced Controller
Pancino and Saez‐Cirion. Immunological Reviews. 2013
Long-term control of infection
Optimal T cell response
CTL
Preserve immune functionLimit viral reservoirsLow dose infection
Early treatment
Cell restriction Innate responses (NK/pDC)
Reduced dynamics of viral replication